Method and apparatus for continuous liquid flow in microscale channels using pressure injection, wicking, and electrokinetic injection

ABSTRACT

Apparatus and methods for modulating flow rates in microfluidic devices are provided. The methods involve modulating downstream pressure in the device to change the flow rate of materials in an upstream region of the device. Such methods include electrokinetic injection or withdrawal of materials through a side channel and the use of an absorbent material to induce wicking in the channel system. The apparatus provided includes a prefabricated wick in the device to provide for flow rate control. Additional methods for determining velocity of a particle and cell incubation time are also provided.

BACKGROUND OF THE INVENTION

[0001] Microfluidic devices have been designed that are useful inperforming high throughput assays useful for biological and chemicalscreening experiments. Both glass and polymer microfluidic devicescomprising microfluidic channels and microfluidic wells are nowavailable. For example, polymer microfluidic devices are provided in PCTapplication WO 98/46438, “Controlled Fluid Transport in MicrofabricatedPolymeric Substrates,” by Parce et al., and glass devices are set forthin a number of publications and patents set forth herein.

[0002] Continuous flow microfluidic systems are set forth in, e.g.,published PCT application WO 98/00231, by Parce et al. These devices areuseful, for example, in screening large numbers of different compoundsfor their effects on a variety of chemical and biochemical systems. Thedevices include a series of channels fabricated on or within thedevices. The devices also can include reservoirs, fluidly connected tothe channels, that can be used to introduce a number of test compoundsinto the sample channels and thus perform the assays. Interfacingmechanisms, such as electropipettors, can be incorporated into thesehigh-throughput systems for transporting samples into wells ormicrofluidic channels. See, e.g., “Electropipettor and CompensationMeans for Electrophoretic Bias,” U.S. Pat. No. 5,799,868, by Parce etal.

[0003] Microfluidic systems for fast, accurate and low costelectrophoretic analysis of materials in the fields of chemistry,biochemistry, biotechnology, molecular biology and numerous otherfields, are described in U.S. Pat. No. 5,699,157 by Parce et al.Techniques for transporting materials through microfluidic channelsusing electrokinetic forces were described in “Electropipettor andCompensation Means for Electrophoretic Bias,” U.S. Pat. No. 5,799,868,by Parce et al.

[0004] Movement of material through microfluidic channels was furtherdescribed in “Variable Control of Electroosmotic and/or ElectrophoreticForces within a Fluid Containing Structure Via Electrical Forces,” U.S.Pat. No. 5,800,690, by Chow et al. In this patent, various powersupplies, such as a time-multiplexed power supplies that vary thevoltage on the system, are described that are used to provide controlover the fluid movement in a microfluidic device.

[0005] Electroosmotic pressure flow has also been described to provideother ways to modulate microfluidic flow rates. For example, thesemethods can involve providing an effective zwitterionic compound in thefluid containing the material to be transported. See, e.g., PublishedPCT application, WO 98/45929 by Nikiforov at el. Additionally, PublishedPCT application, WO 98/56956 by Kopf-Sill et al. provides methods ofcorrecting for variable velocity in microfluidic devices.

[0006] Channel dimensions have also been varied to provide furthercontrol over the movement of fluid through the channels, such as in“Microfluidic Systems Incorporating Varied Channel Dimensions,” See,e.g., U.S. Pat. No. 5,842,787, by Kopf-Sill et al.

[0007] Although corrections can be made for variable velocities, see,e.g., Published PCT application, WO 98/56956, by Kopf-Sill et al., it isadvantageous to be able to rapidly and easily modulate the velocity orflow rate of a component in a microfluidic device. There exists a needfor high throughput screening methods, and associated equipment anddevices, that are capable of performing repeated, accurate assay,operating at very small volumes and at regulated and/or continuous flowrates. These assays are particularly useful for high throughputscreening, as well as for a variety of research applications.

[0008] The present invention meets these and a variety of other needs.In particular, the present invention provides novel methods andapparatuses for performing assays with continuous or discontinuous flowrates, as well as other apparatus methods and integrated systems, whichwill be apparent upon complete review of the disclosure.

SUMMARY OF THE INVENTION

[0009] This invention provides methods, devices and systems forsustaining and/or modulating and/or measuring flow rates in amicrofluidic system by modulating pressure downstream from the region ormaterial of interest. In accordance with the invention, flow rates aremodulated or regulated to provide continuous or discontinuous flow by avariety of means. For example, an absorbent material such as anabsorbent gel, absorbent polymer material or cellulose containingmaterial is optionally placed downstream from the region or material ofinterest. Alternatively, or additionally, electrokinetic or pressurebased injection or withdrawal of materials into or from the systemdownstream of the material or region of interest may be used to modulateupstream flow rates. For example, a wick (which can be pre-wetted, dryor wetted in position in contact with a microfluidic system) can act bycapillary action to draw material through channels or wells in which itis placed in fluidic contact. Alternatively, or additionally, a volumeof liquid is optionally injected or withdrawn downstream of the materialor region of interest and the flow rate modulated by creating a pressuredifferential at the site of injection. Microfluidic devices are providedthat contain absorbent materials in particular wells or that haveparticular wells located to serve as microfluidic injection sites.

[0010] In one embodiment, the invention provides a method of modulatingthe flow rate of material in a microfluidic channel system by modulatingpressure downstream of the material, thereby increasing or decreasingflow rate of the material in the channel. Pressure modulation isoptionally achieved by placing an absorbent material, such as a wick, ina microfluidic well, by electrokinetic injection, by creating a pressuredifferential, or by a combination of these three methods.

[0011] The absorbent material used to modulate pressure in amicrofluidic system is placed. e.g., within a well, such as a wastewell, or at the junction of a well and a channel. It can extend beyondthe top of the well or remain within the well. The absorbent materialis, e.g., a solid, porous, gel, or polymeric material. It is optionally,e.g., a high salt fluid, a thermoplastic polymer (e.g., which is porousor sintered) a porous plastic, or a polyolefin resin. Typically, theabsorbent material will be a cellulosic material such as a piece ofpaper, e.g., a Kimwipe, paper towel, cellulose membrane, nylon membrane,Whatman™ filter, blotting paper, filter paper, cloth or fibrousmaterial, or a polymer, such as dried cross-linked polyacrylamide, or aporous or sintered polymer such as a porous or scintered polyethylene,polypropylene, polyvinylidene fluoride, ethylene-vinyl acetate,polytetrafluoroethylene, stryene-acrylonitrile, polysulfone,polycarbonate, or polyhthalate polymer.

[0012] The invention also provides a method for modulating flow rate ofmaterial in a microfluidic system by electrokinetic injection of asecond material downstream of the first material or region of interest.The flow rate is monitored before injection and/or after injection sothat it is sustained at a certain level and controlled.

[0013] The invention provides methods of monitoring flow rates bydetecting a signal from the material in the channel and measuring theduration and amplitude of a signal that is detected by monitoringfluorescence, phosphorescence, radioactivity, pH, or charge.

[0014] In another embodiment, the invention provides a method fordetermining velocity of a particle in a microfluidic channel system bydetecting a signal from the particle for a period of time. The signalamplitude corresponds to the number of particles, and the durationcorresponds to the velocity of the particle. Once determined, thevelocity is optionally modulated or made constant by electrokineticinjection or by use of an absorbent material such as a wick.

[0015] In another embodiment, the invention provides for microfluidicdevices that contain wicks or other absorbent materials for use inmodulating the flow rate of materials in the device. The devices aremade to accommodate flow rate control by wicking or other capillaryforces, as described above, by electrokinetic injection, pressuredifferential or a combination of flow rate control elements. Amicrofluidic system optionally includes a computer and software forsimultaneous or sequential monitoring or control over flow rates, aswell as analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic drawing of a microfluidic device of thepresent invention.

[0017]FIG. 2 is a schematic drawing of an alternate device of theinvention.

[0018]FIG. 3 (panels A, B and C) are schematic drawings of a devicecomprising an absorbent material such as a wick.

[0019]FIG. 4 is a graph showing that as the current is increased for aside-chamber electrokinetic injection into the main chamber, dye in themain chamber is diluted, thereby demonstrating that an effect of theside channel injection is to change the main channel flow rate.

[0020]FIG. 5 is a graph showing that toggling an electrokineticinjection current on and off rapidly changes the dilution of a dye inthe main chamber, indicating rapid control of flow from a side channel.Time is shown in seconds on the graph and the signal is recorded inrelative fluorescence units.

[0021]FIG. 6 is a graph showing the effect of buffer injections from theside channel on the flow rate of cells in the main channel.

[0022]FIG. 7 is a graph of live cells detected using fluorescent DNA dyeas they flow through a microfluidic device. The cells are detected asthey flow past a fluorescent reader. Each peak represents a cell ormultiple cells depending on how many are in the reading area at once. Anabsorbent material such as a wick was placed into the device at 400seconds.

[0023]FIG. 8 is a graph of the cells flowing through a microfluidicdevice after the wick was removed at 500 seconds.

[0024]FIG. 9 is a graph of the cells as they flow through a device whichdoes not have a wick in fluidic contact with fluid in the device.

[0025]FIG. 10 shows the cells as they stop flowing through a chamber,i.e., with no wick in place, e.g., at about 1000 seconds, or about 500seconds after the wick was removed.

[0026]FIG. 11 is a graph showing cells flowing through a device after awick was replaced at 1300 seconds.

[0027]FIG. 12 is a graph showing that the cells continued to flowthrough the device with a wick in place until at least 2700 seconds, atwhich time the cells still maintained a high flow rate, evidenced by thenarrow peak widths.

[0028]FIG. 13 shows graphs from a calcium flux assay performed using thedevices of the invention.

DEFINITIONS

[0029] “Microfluidic,” as used herein, refers to a system or devicehaving fluidic conduits that are generally fabricated at the micron tosubmicron scale, e.g., typically having at least one cross-sectionaldimension in the range of from about 0.1 μm to about 500 μm. Themicrofluidic system of the invention is fabricated from materials thatare compatible with the conditions present in the particular experimentof interest. Such conditions include, but are not limited to, pH,temperature, ionic concentration, pressure, and application ofelectrical fields. The materials of the device are also chosen for theirinertness to components of the experiment to be carried out in thedevice. Such materials include, but are not limited to, glass, quartz,silicon, and polymeric substrates, e.g., plastics, depending on theintended application.

[0030] As used herein, “channel” refers to a fluidic conduit. Channelsoptionally connect with wells, other channels, or other features of amicrofluidic device. The channels are typically of microfluidicdimensions as discussed above.

[0031] The term “downstream” refers to a location in a channel that isfarther along the channel in a selected direction of fluid or materialflow, relative to a selected site or region.

[0032] A “well” typically refers to a chamber or reservoir in amicrofluidic device or system, e.g., for adding or removing a componentto or from the system. The well is optionally open topped or closedwithin the body of the device. A “waste well” is that chamber to whichthe results or remains of an experiment are directed. Waste products ofan experiment are optionally collected and/or removed from the wastewell. A well also optionally functions as a port for providing access tochannels, e.g., electrical or fluidic access.

[0033] A “wick,” as used herein, refers to an absorbent material used tomodulate continuous flow in a microfluidic system. Typically, the wickwill comprise an absorbent material which absorbs a fluid such as anaqueous or non-aqueous solution. The wick is optionally the same size asthe well or other microfluidic element in which it is contained, smallerthan the well, larger than the well, extending beyond the upper edge ofthe well, or in any other configuration.

[0034] An “absorbent material” is a substance that has the power orcapacity or tendency to absorb or take up fluid. Absorption mechanismsinclude capillary forces, osmotic forces, solvent or chemical action, orthe like. The absorbent material of the invention is optionally a solidmaterial, a porous material, a sintered material, a gel, a polymer, ahigh salt fluid, a thermoplastic polymer (such as any Porex™ polymermaterial), a polyolefin resin, or a porous plastic (including, e.g.,Porex™ plastics). The absorbent material can be cellulosic material suchas paper (e.g., a piece of Kimwipe™, paper towel or the like), but isoptionally dried cross-linked polyacrylamide, agarose, or a porous orsintered polymer (e.g., such as a porous or sintered polyethylene,polypropylene, high molecular weight polyethylene, polyvinylidenefluoride, ethylene-vinyl acetate, polytetrafluoroethylene,stryene-acrylonitrile, polysulfone, polycarbonate, dry sephadex,dextran, or polyhthalate), or other materials which will be apparentupon complete review of this disclosure. Additionally, an absorbentmaterial can be a combination of one or more of the above materials.

[0035] A “junction” or “intersection” between two channels or between achannel and a well refers to a region in which two or more channels orwells are in fluid communication with each other. The term encompasses“T” intersections, cross intersections, “wagon wheel” intersections ofmultiple channels and/or wells, or any other channel/well geometry wheretwo or more channels and/or wells are in such fluid communication.

[0036] As used herein, the term “thermoplastic polymer” refers toplastics and synthetic resins that are remelted and cooled withoutundergoing any appreciable chemical changes, such as cellulose acetateand e.g., a variety of pouros or sintered polymers made by PorexTechnologies as well as a variety of other commercial sources. See,Porex Technologies catalog, Fairburn, Ga. Other commercial sourcesinclude, e.g., Sigma and Aldrich. It includes, but is not limited to,porous or scintered polymers or polymer or plastic particles made from,e.g., ultra high density polyethylene, polypropylene, high molecularweight polyethylene, polyvinylidene fluoride, ethylene-vinyl acetate,polytetrafluoroethylene, stryene-acrylonitrile, polysulfone,polycarbonate, and polyhthalate. A variety of thermoplastic polymers aredescribed in the Kirk-Othmer Encyclopedia of Chemical Technology, 4_(th)Edition, Wiley Interscience.

[0037] “Porous plastic” refers to a plastic material that is full ofholes or pores, or that is capable of absorbing moisture, or which ispermeable by liquids. These materials include, but are not limited to, apolyethylene particle, a polypropylene particle, a high molecular weightpolyethylene particle, a polyvinylidene fluoride particle, anethylene-vinyl acetate particle, a polytetrafluoroethylene particle, astryene-acrylonitrile particle, a polysulfone particle, a polycarbonateparticle, and a polyhthalate particle, e.g., such as scintered orplastic beads made by Porex Technologies from polyolefin resins or asavailable from other commercial sources, such as Sigma and Aldrich.

[0038] The functioning of the system is indicated by the production of adetectable event or signal. “Detection” is accomplished by monitoringsignals such as optically detectable chromophoric or fluorescent signalsthat are associated with the functioning of the particular model systemused. Other detection systems are described supra, and in citedreferences.

[0039] As used herein, the term “continuous flow” generally refers to anunbroken or contiguous stream of the particular material or compositionthat is being continuously flowed. For example, a continuous flow of asample includes a constant or variable fluid flow having a set velocity,or alternatively, a fluid flow which includes pauses in the flow rate ofthe overall system, such that the pause does not otherwise interrupt theflow stream.

[0040] “Velocity” typically refers to the distance a selected componenttravels (l) divided by the time (t) required for the travel. In manyembodiments, the velocity under consideration is essentially constant,e.g., for the travel of reaction components along the length of amicrochannel under a constant rate of current in an electrokineticsystem or under a constant applied pressure differential. See, e.g.,Published PCT application, WO 98/56956, by Kopf-Sill et al. for adiscussion of variable velocity in microfluidic systems. Where thevelocity changes significantly over time, due, e.g. to change of appliedcurrent in an electrokinetic system, or where a change from substrate toproduct results in a slow acceleration (or deceleration) in the system,an “instantaneous velocity” equal to the change in distance for aselected time (Δ1/Δt) can be determined by graphing distance againsttime and taking the tangent of the resulting function at a particularpoint in time.

DETAILED DESCRIPTION

[0041] Microfluidic devices have been used in biochemical fields toperform high throughput screening assays. One problem in the use of thedevices in assays is ensuring a constant and continuous flow rate. Flowin microfludic systems are typically powered by a pressure based systemor electrokinetic fluid direction systems. Problems encountered includea cessation or decrease in the flow rate when capillary action issuspended due to evaporation from the waste well or sample materialadhering to the corners of the channels. However, for bioassay systems,a constant flow of materials is useful to maintain the assay, ascertaincell incubation time, and reduce time for multiple screening assays. Inaddition, the ability to modulate flow rates is equally useful inmicrofluidic systems.

[0042] The present invention provides methods for achieving continuousand consistent flow in a microfluidic device by modulation of pressuredownstream from any fluid flow that requires regulation as well as, moregenerally, modulating flow of materials in channels. One way the flowrate is modulated is by positioning an absorbent material in a well orwaste well of the device. A wick is one such absorbent material. Devicesthat contain the elements necessary to perform such regulation are alsodescribed in the present invention, e.g., devices that contain wicks.

[0043] An alternative or additional way the flow rate is modulated is byelectrokinetic or pressure based injection or withdrawal downstream ofthe channel region containing the sample stream to be modulated.

[0044] In addition to providing methods for regulating or modulatingflow rates or achieving a continuous flow rate, the invention alsoprovides methods for monitoring and detecting the flow rate in amicrofluidic system and measuring the velocity of a particle, such as abead or cell, in a bioassay carried out in a microfluidic system.

[0045] I. Methods of Modulating Flow Rate in a Microfluidic System

[0046] The flowing of materials, such as a suspension of cells, throughthe channels of a microfluidic device is carried out by a number ofmechanisms, including pressure based flow, electrokinetic flow, ormechanisms that utilize a hybrid of the two. As noted above, continuousflow is desirable in certain applications, e.g., to modulate or controlincubation times. The present invention provides methods of achievingcontinuous flow and/or regulating or modulating flow rates, e.g.,controllably changing the flow rate, in a microfluidic device bymodulating the pressure downstream from the sample or material ofinterest.

[0047] A. Sustained Flow is Achieved by Modulating Downstream Pressure

[0048] Flow rates through a channel may vary as the assay progresses.For example, material may stick or adhere to the walls of the channel orwell and thereby reduce capillary action and/or mask the surface chargefor electrokinetic purposes and slow the flow rate of the materialthrough the channel. Alternatively, evaporation from the waste well mayconcentrate salts in the fluid in the channel, thereby increasing thedensity and viscosity of the fluid and decreasing the flow rate. In manyapplications, however, a known and/or constant flow rate is useful, forexample, when attempting to establish the incubation time of a cell anda test compound. Furthermore, continuous and/or constant flowfacilitates high throughput screening.

[0049] Additionally, electrokinetic forces are sometimes avoided toprevent leakage of dyes and non-specific cell responses at highvoltages. In these instances, a constant flow is achieved by modulationof the pressure downstream from the sample to be analyzed, for example,by use of a wick.

[0050] In these embodiments, a constant flow rate is achieved or to flowregulated by placing an absorbent material in a well channel orreservoir of the microfluidic system. This absorbent material absorbsand draws fluid through the channel. By drawing the fluid up and out ofthe well, the flow rate stabilizes and is not affected by the adhesionof material to corners, which decreases capillary forces. Likewise, thewick can draw the liquid up before evaporation and thereby avoidconcentrating the material and making it denser. The salts of the liquidwill likewise be drawn up by the wick along with the fluid material. Thewick material, size, shape and placement are optionally varied toachieve the desired flow rate.

[0051] Alternatively, the pressure may be regulated by fluiddisplacement, e.g., using a piston, pressure diaphragm or probe todisplace liquid and raise or lower the pressure. An alternate way tomodulate the pressure is through a side channel electrokinetic injectionor withdrawal (e.g., downstream from the side where flow is modulated),creating a pressure differential by electrokinetically injecting orwithdrawing another liquid into or from a side channel and therebymodulating the flow rate. This provides a pressure change downstream ofthe material of interest, thereby decreasing or increasing the flowrate. This method can also be easily regulated and adjusted as the assayprogresses, making it useful for achieving continuous flow, or generallyfor modulating flow rates.

[0052] B. Electrokinetic and Pressure Based Transport Systems.

[0053] One method of achieving transport or movement of samples throughmicrofluidic channels is by electrokinetic material transport, whichforms the basis of the electrokinetic injection or withdrawal methods ofthe present invention. “Electrokinetic material transport systems,” asused herein, includes systems that transport and direct materials withinan interconnected channel and/or chamber containing structure, throughthe application of electrical fields to the materials, thereby causingmaterial movement through and among the channel and/or chambers, i.e.,cations will move toward a negative electrode, while anions will movetoward a positive electrode.

[0054] Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. For electrophoretic applications, the walls ofinterior channels of the electrokinetic transport system are optionallycharged or uncharged. Typical electrokinetic transport systems are madeof glass, charged polymers, and uncharged polymers. The interiorchannels are optionally coated with a material which alters the surfacecharge of the channel.

[0055] Other electrokinetic material direction and transport systemsrely upon the electroosmotic flow of fluid and material within a channelor chamber structure which results from the application of an electricfield across such structures. In brief, when an appropriate fluid isplaced in a channel or other fluid conduit having functional groupspresent at the surface, those groups ionize. For example, where thesurface of the channel includes hydroxyl functional groups at thesurface, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface will possess a net negativecharge, whereas the fluid will possess an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid. By applying an electric field along the length ofthe channel, cations will flow toward the negative electrode. Movementof the positively charged species in the fluid pulls the solvent withthem.

[0056] An electrokinetic device moves components by applying an electricfield to the components in a microfluidic channel, such as first channelregion 115 in FIG. 1. By applying an electric field along the length ofthe channel, cations will flow toward a negative electrode, while anionswill flow towards a positive electrode. Movement of charged species inthe fluid pulls the solvent with the fluid, provided the fluid ismobile. In pure electrophoretic applications, elements of the fluid arenot mobile, e.g., due to cross-linking, i.e., where the fluid is a gelmatrix, or due to a lack of surface charge on the walls of the interiorchannel.

[0057] The steady state velocity of fluid movement is generally given bythe equation: $v = \frac{{ɛ\xi}\quad E}{4{\pi\eta}}$

[0058] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. The solvent velocity is,therefore, directly proportional to the surface potential. In thisinvention, electrokinetic forces are used to modulate the velocity ofmaterials in the channels of a microfluidic device.

[0059] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, sequentially or, more typically, simultaneously, to eachof the reservoirs, including ground. Such a voltage controller isimplemented using multiple voltage dividers and multiple relays toobtain the selectable voltage levels. Alternatively, multipleindependent voltage sources are used. The voltage controller iselectrically connected to each of the reservoirs via an electrodepositioned or fabricated within each of the plurality of reservoirs. Inone embodiment, multiple electrodes are positioned to provide forswitching of the electric field direction in a microchannel, therebycausing the analytes to travel a longer distance than the physicallength of the microchannel. Use of electrokinetic transport to controlmaterial movement in interconnected channel structures was described,e.g., in WO 96/04547 to Ramsey, which is incorporated by reference.

[0060] Modulating voltages are concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous or discontinuous (e.g., a regularly pulsed field causing thesample to oscillate direction of travel) flow of labeled componentstoward a waste reservoir. Particularly, modulation of the voltagesapplied at the various reservoirs can move and direct fluid flow throughthe interconnected channel structure of the device.

[0061] Some biological cell assays useful in the present invention donot work well in an electrically controlled system because high voltagesmay cause an undesired cellular response. Another way to control flowrates is through creation of a pressure differential. For example, in asimple passive aspect, a cell suspension is deposited in a reservoir orwell at one end of the channel, and at sufficient volume or depth, thatthe cell suspension creates a hydrostatic pressure differential alongthe length of the channel, e.g., by virtue of its having greater depththan a well at an opposite terminus of the channel. Typically, thereservoir volume is quite large in comparison to the volume or flowthrough rate of the channel, i.e., 10 μl reservoirs or larger ascompared to a 100 μm channel cross section. Another pressure basedsystem is one that displaces fluid in a microfluidic channel using,e.g., a probe, piston, or pressure diaphragm.

[0062] Alternatively, a pressure differential is applied across thelength of the channel. For example, a pressure source is optionallyapplied to one end of the channel, and the applied pressure forces thematerial through the channel. For example, in FIG. 1, a pressure appliedat main injection well 110 or first channel region 115 would force acell suspension through reading area 120, second channel region 125, andinto waste well 130. The pressure is optionally pneumatic, e.g., apressurized gas or liquid, or alternatively a positive displacementmechanism, i.e., a plunger fitted into a material reservoir, for forcingthe material along through the channel. Pressure can, of course, also bedue to electrokinetic force.

[0063] Alternatively, a vacuum source (i.e., a negative pressure source)is applied to a reservoir or well at the opposite end of the channel todraw the suspension through the channel. In FIG. 1, a vacuum sourceplaced in waste well 130 draws a cell suspension from, e.g., maininjection well 110, or from buffer well 145 or from reading area 120.Pressure or vacuum sources are optionally supplied external to thedevice or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel. Examples of microfabricated pumps havebeen widely described in the art. See, e.g., published InternationalApplication No. WO 97/02357.

[0064] In screening applications, varying the flow rate of a cellsuspension is optionally used, e.g., to vary the incubation time of thecells with a test compound (e.g., potential inhibitor, activator, ligandor the like). In particular, by slowing the flow rate of cells along thechannel, one can effectively lengthen the amount of time betweenintroduction of a test compound and detection of its effects. Channellengths, detection points, or test compound introduction points arevaried in the fabrication of the microfluidic device to vary incubationtimes. However, this invention provides easier and more flexible ways tovary and regulate the flow rate in a channel, thereby providing betterways to monitor and control cell incubation time. The pressure basedelements and electrokinetic transport systems discussed above are usedwith this invention to provide continuous flow rates.

[0065] II. Using a Wick or Other Absorbent Material in a MicrofluidicDevice to Control the Flow Rate

[0066] A. Wick/Absorbent Materials

[0067] A wick is an absorbent material used to modulate and sustain flowrates of a sample in a microfluidic system. Typically the wick willcomprise an absorbent material, i.e., a substance that has the power,capacity or tendency to absorb or take up fluid. Absorption mechanismsinclude capillary forces, osmotic forces, solvent or chemical action orthe like.

[0068] Absorbent materials of the invention include solid materials,porous materials, gels, porous or sintered polymers, high salt fluids,thermoplastic polymers (such as those available from Sigma, Aldrich,Porex™, etc.), polyolefin resins, or porous plastics, including, e.g.,Porex™ plastics.

[0069] The absorbent wick material is optionally a cellulosic materialsuch as paper, e.g., Kimwipe™, paper towel or the like. The absorbentmaterial can also be, e.g., dried polyacrylamide, polyethylene,polypropylene, a high molecular weight polyethylene, polyvinylidenefluoride, ethylene-vinyl acetate, polytetrafluoroethylene,stryene-acrylonitrile, polysulfone, polycarbonate, dextran, drysephadex, or polyhthalate, or other materials which will be apparentupon complete review of this disclosure. The absorbent material can bewetted prior to being placed into contact with the microfluidic device,or can be dry prior to placement in contact with a microfluidic device.Pre-wetting can aid in establishing capillary flow for some materials,but is not typically required. For example, one can fill a device withbuffer, add samples and e.g., cells or other biological materials, applya wick or other absorbent material in fluid communication with fluid inthe device, and even reduce evaporative effects by applying a cap to thewick to prevent evaporation. One of skill can easily assess thedesirablity of pre-wetting by varying the wetting strategy and observingany resulting alteration in flow properties.

[0070] The absorbent wick material is optionally a disposable orreusable material, such as a piece of Kimwipe™, or other absorbentcellulosic material, or, e.g. a porous plastic plug that fits into amicro well, or the like. Additionally, the absorbent material may takeon a variety of shapes. It is optionally a narrow rectangular piece ofabsorbent material that extends beyond the upper edge of a well or intothe channel or a rounded piece of absorbent material that sits insidethe well. More of the absorbent material is optionally situated abovethe fluid surface than below or the reverse, depending on the flow rateone wants to achieve. Alternatively, the wick is optionally a solid plugof absorbent material that fits snugly or loosely in a well or reservoir(or channel). It can also be a porous plastic tube that extends into achannel or extends beyond the top of the well or reservoir in which itis located. Almost any shape imaginable is optionally used as a “wick”and its effect on the flow rate is easily determined. For example, theflow rate is optionally determined by monitoring the amount of labeledor otherwise detectable material passing the detection window, e.g.,using a microscope. For example, using this monitoring technique, it wasdetermined that a narrow wick provides a slower flow rate than a widewick.

[0071] The wick or other absorbent material used optionally includes asurfactant to assist the wicking process. Surfactants can be obtainedfrom any of a variety of sources, such as the SIGMA chemical company(Saint Louis, MO). The absorbent material of the present invention isoptionally soaked in a surfactant prior to use or before, during orafter fabrication.. For example, such surfactant impregnated materialsinclude, but are not limited to, the products manufactured by FiltronaRichmond Inc., Richmond, Va.

[0072] B. Location of the Wick and Use of the Wick Within theMicrofluidic System

[0073] A wick is optionally used in a microfluidic device by positioningit in a well, such as a waste well, or at the junction between a welland a channel. The wick need only be placed at a location that allows itto take up fluid and pull the material or sample stream toward it.

[0074] The wick is optionally internal or external to the microfluidicdevice or system. For example, the wick is optionally placed entirelywithin a well or channel of a microfluidic system, or it can extend outfrom the top of a well or reservoir. Furthermore, the wick is optionallythe same size as the well in which it is contained, smaller than thewell, or larger than the well, in which case it may extend beyond thetop of the well.

[0075] In one embodiment, a wick is used in a microfluidic channel byfirst filling the channels with a liquid and designating an empty wellas the “waste well”. A piece of absorbent material is sized to fit atleast partially in the well (or, e.g., a capillary can extend from thewell to the wick). The wick is placed into the well and optionallywetted with liquid to help begin the wicking action by drawing theliquid up and out of the well (generally, the absorbent material can bepre-wetted (i.e., wetted prior to contact with the fluid, reservoir orchannel at issue) to facilitate or regulate osmotic pressure and,therefore, wicking action. Furthermore, by placing a wick in one well,the entire system of channels or any subset thereof is optionallyregulated by fluidly connecting all or some channels to the wellcontaining the wick.

[0076] C. Devices and Systems That Contain Wicks

[0077] In other embodiments, the wick is a disposable or re-usablecellulosic material, e.g., piece of paper, membrane, filter, fabric, orother fibrous material that is replaced after each use of the wick. Inan alternate embodiment, it is a reusable piece of porous plastic thatis placed in fluidic contact with a fluidic channel in the microfluidicsystem. Microfluidic devices and systems are fabricated, as describedin, e.g., U.S. Pat. No. 5,842,787, titled “Microfluidic SystemsIncorporating Varied Channel Dimensions,” by Kopf-Sill et al., or in theother references herein, with an absorbent material or wick beingfabricated into a well of the device. The wick is positioned in the wellor at the junction between a well and a channel. The wicks placed intofabricated devices are made from the materials, used and positioned asdescribed above. The wicks or other absorbent materials are optionallymanufactured and/or sold separately and fitted into the well or wells ofprefabricated microfluidic devices. An alternative way to fabricate amicrofluidic device with a wick is to use an absorbent material that issprayed into the well, such as an aerosol particulate spray (e.g.,comprising porous particulate matter).

[0078] III. Electrokinetic Injection or Withdrawal to Modulate the FlowRate in a Microfluidic System

[0079] In the event that the absorbent material does not provide theflow rate desired, an additional or substitute method of modulation isoptionally used. Electrokinetic material transport, as described above,is optionally used to inject fluids or other materials into the regionof interest in a microfluidic channel. Electrokinetic injection ofmaterials into a microfluidic device is accomplished by providing avoltage gradient between the source of test materials, i.e., a well orreservoir, and the intersecting channel structure in the interior of thedevice. The voltage is applied such that the material flows from thewell into a channel or from a channel into a well. This voltage isoptionally applied by a power source, for example, via electrodes.Furthermore, this method can be applied in conjunction with the wickmethod described above to provide a full range of continuous flow rates.

[0080] Upstream flow modulation is achieved by decreasing the upstreamflow rate, e.g., by flowing liquid into the channel downstream orincreasing the upstream flow rate by drawing liquid out of the channeldownstream of the region of interest. Changing the channel geometry, forexample by increasing or decreasing channel width, or by etching anetwork of capillaries downstream of a selected channel region serves asimilar function, but this is not conveniently changed once the devicehas been fabricated, and does not offer the same flexibility aselectrokinetic or pressure based injection. Nevertheless, the use ofselected channel geometries, e.g., for performing repetitive assays isalso useful. For example, etching a plurality of capillary channelsdownstream of a selected channel region (typically during devicemanufacture), where the plurality of channel regions are, during use ofthe apparatus, in fluidic communication with the selected channelregion, also finds use in the present invention.

[0081] Fluidic injection provides flexibility because injection ratescan be adjusted as the need for modulation arises or changes, while achange of channel geometry is typically permanent once the device hasbeen fabricated. Electrokinetic injection provides an adjustable flowrate control by moving liquid into the main channel through aside-channel injection, e.g., through the application of an appropriatecurrent between the side channel and main channel. The higher the sidechannel injection rate, the slower the material (e.g., cell) movementupstream of the injection site. When the side channel injection isturned off or decreased, the material flow rate upstream of theinjection site increases. Alternatively, the polarity of theelectrokinetic injection can be reversed to increase the flow rate inthe main channel by pulling fluid out of the main channel into the sidechannel.

[0082] Furthermore, because injection is optionally performed in a sidechannel downstream of the detection window, the buffer used does notnecessarily sample contact materials such as cells in an assay in themain channel until after the results of the assay have been measured.Therefore, the velocity of materials is controlled without moving partsand the pumped buffer can be optimized for pumping efficiency, since itwill not affect the materials in the assay.

[0083] In another embodiment, the two above methods of flow modulationare combined. For example, a wick is used to provide sustained flow at ahigh rate and if a slower rate is needed the electrokinetic injectionmethod is used to slow the rate down to a desired level. An addedadvantage is that both are easily and readily adjusted to suit the needsof the assay of interest.

[0084] IV. Methods for the Monitoring of Flow Rate in a MicrofluidicDevice and the Detection of Bioassay Signals

[0085] This invention provides methods of monitoring samples in amicrofluidic device. First, a sample must be introduced (e.g., injected,flowed or placed) into the device. Typically, samples are injected intoa channel, well or reservoir, for example, using a micro-pipettor orelectropipettor. Once injected, the sample is transported through thechannels of the device and modulated by one of the methods describedabove, such as electrokinetic forces, pressure based elements, wicks, orcombinations thereof.

[0086] Once a sample has been introduced into the device and is beingtransported through the channel or channels of the device, the flow rateof the sample and the velocity of a particle in the sample, such as acell, are measured. To monitor the sample and measure the flow rate orvelocity, first the sample or sample components are detected. Detectiontypically occurs through the use of a label associated with the materialof interest. A “label” is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention includefluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P,³³P, etc.), enzymes (e.g., horse-radish peroxidase, alkaline phosphataseetc.) calorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The labelis coupled directly or indirectly to a component of the assay accordingto methods well known in the art. As indicated above, a wide variety oflabels are used, with the choice of label depending on sensitivityrequired, ease of conjugation with the compound, stability requirements,available instrumentation, and disposal provisions. Non-radioactivelabels are often attached by indirect means. Generally, a ligandmolecule (e.g., biotin) is covalently bound to the molecule. The ligandthen binds to an anti-ligand (e.g., streptavidin) molecule which iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands areoptionally used. Where a ligand has a natural anti-ligand, for example,biotin, thyroxine, or cortisol, it is used in conjunction with thelabeled, naturally occurring anti-ligands. Alternatively, any haptenicor antigenic compound can be used in combination with an antibody (see,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, N.Y. for a general discussion of how to and useantibodies). The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include, e.g., luciferin,and 2,3-dihydrophthalazinediones, e.g., luminol.

[0087] In some embodiments, a first and second label on the same ordifferent components interact when in proximity (e.g., due tofluorescence resonance energy transfer or “FRET”), and the relativeproximity of the first and second labels is determined by measuring achange in the intrinsic fluorescence of the first or second label. Forexample, the emission of a first label is sometimes quenched byproximity of the second label. This technique is particularly suited formeasurement of binding reactions, protein-protein interactions and otherbiological events altering the proximity of two labeled molecules. Manyappropriate interactive labels are known. For example, fluorescentlabels, dyes, enzymatic labels, and antibody labels are all appropriate.Examples of interactive fluorescent label pairs include terbium chelateand TRITC (tetrarhodamine isothiocyanate), europium cryptate andAllophycocyanin, DABCYL and EDANS and many others known to one of skill.Similarly, two colorimetric labels can result in combinations whichyield a third color, e.g., a blue emission in proximity to a yellowemission provides an observed green emission. With regard to preferredfluorescent pairs, there are a number of fluorophores which are known toquench one another. Fluorescence quenching is a bimolecular process thatreduces the fluorescence quantum yield, typically without changing thefluorescence emission spectrum. Quenching can result from transientexcited state interactions, (collisional quenching) or, e.g., from theformation of nonfluorescent ground state species. Self quenching is thequenching of one fluorophore by another; it tends to occur when highconcentrations, labeling densities, or proximity of labels occurs. FRETis a distance dependent excited state interaction in which emission ofone fluorophore is coupled to the excitation of another which is inproximity (close enough for an observable change in emissions to occur).Some excited fluorophores interact to form excimers, which are excitedstate dimers that exhibit altered emission spectra (e.g., phospholipidanalogs with pyrene sn-2 acyl chains); see, Haugland (996) Handbook ofFluorescent Probes and Research Chemicals Published by Molecular Probes,Inc., Eugene, Oreg. e.g., at chapter 13).

[0088] Detectors for detecting the labeled compounds of the inventionare known to those of skill in the art. Thus, for example, where thelabel is a radioactive label, means for detection include ascintillation counter or photographic film as in autoradiography. Wherethe label is a fluorescent label, it is detected by exciting thefluorochrome with the appropriate wavelength of light and detecting theresulting fluorescence. The fluorescence is optionally detectedvisually, by means of photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers,phototubes, photodiodes or the like. Similarly, enzymatic labels aredetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels are detected simply by observing the color associated with thelabel. This is done using a spectrographic device, e.g., having anappropriate grating, filter or the like allowing passage of a particularwavelength of light, and a photodiode, or other detector for convertinglight to an electronic signal, or for enhancing visual detection.

[0089] The microfluidic device includes a detection window or zone atwhich a signal is monitored. For example, reactants or assay componentsare contacted in a microfluidic channel in first region 115, andsubsequently flowed into reading area 120, comprising a detection windowor region. The first and second channel region are optionally part of asingle channel, but can also be separate channels, e.g., which are influid connection. This detection window or region typically includes alight or radiation transparent cover allowing visual or opticalobservation and detection of the assay results, e.g., observation of acolorometric, fluorometric or radioactive response, or a change in thevelocity of colorometric, fluorometric or radioactive component.Detectors detect a labeled compound. Example detectors includespectrophotometers, photodiodes, microscopes, scintillation counters,cameras, film and the like, as well as combinations thereof. Examples ofsuitable detectors are widely available from a variety of commercialsources known to persons of skill.

[0090] In one aspect, monitoring of the signals at the detection windowis achieved using an optical detection system. For example, fluorescencebased signals are typically monitored using, e.g., in laser activatedfluorescence detection systems which employ a laser light source at anappropriate wavelength for activating the fluorescent indicator withinthe system. Fluorescence is then detected using an appropriate detectorelement, e.g., a photomultiplier tube (PMT). Similarly, for screensemploying colorometric signals, spectrophotometric detection systems areemployed which detect a light source at the sample and provide ameasurement of absorbance or transmissivity of the sample. See also, ThePhotonics Design and Applications Handbook, books 1, 2, 3 and 4,published annually by Laurin Publishing Co., Berkshire Common, P.O. Box1146, Pittsfield, Mass. for common sources for optical components.

[0091] In alternative aspects, the detection system comprisesnon-optical detectors or sensors for detecting a particularcharacteristic of the system disposed within the detection window. Suchsensors may include temperature (useful, e.g. when a reaction producesor absorbs heat), conductivity, potentiometric (pH, ions), amperometric(for compounds that are oxidized or reduced, e.g., O₂, H₂O₂, I₂,oxidizable/reducible organic compounds, and the like).

[0092] Alternatively, schemes similar to those employed for theenzymatic system are optionally employed, where there is a signal thatreflects the interaction of the receptor with its ligand. For example,pH indicators which indicate pH effects of receptor-ligand binding areoptionally incorporated into the device along with the biochemicalsystem, i.e., in the form of encapsulated cells, whereby slight pHchanges resulting from binding are detected. See Weaver, et al.,Bio/Technology (1988) 6:1084-1089. Additionally, one can monitoractivation of enzymes resulting from receptor ligand binding, e.g.,activation of kinases, or detect conformational changes in such enzymesupon activation, e.g., through incorporation of a fluorophore which isactivated or quenched by the conformational change to the enzyme uponactivation.

[0093] One conventional system carries light from a specimen field to acooled charge-coupled device (CCD) camera. A CCD camera includes anarray of picture elements (pixels). The light from the specimen isimaged on the CCD. Particular pixels corresponding to regions of thesubstrate are sampled to obtain light intensity readings for eachposition. Multiple positions are processed in parallel and the timerequired for inquiring as to the intensity of light from each positionis reduced. Many other suitable detection systems are known to one ofskill.

[0094] Once the sample or its components are detected, the flow rate isoptionally monitored by measuring the amplitude and duration of thedetection signal. The flow rate of the materials being assayed is thendetermined. In one aspect, the number of cells being measured isdetermined by the amplitude of the signal and the flow rate isdetermined by the duration of the signal for single cells. For example,the flow rate equals the length of the detection window or reading framedivided by the duration of the signal; thus, a longer signal durationcorresponds to a slower flow rate.

[0095] V. Determination of Velocity

[0096] The velocity of a particle is optionally determined using themethods of the invention. When a particle, such as a cell in a cellassay is injected into a microfluidic system as described below, itsvelocity is optionally determined and modulated by the methods describedabove. While the discussion below is specific for cells for purposes ofillustration, one of skill will recognize that other components, e.g.,particles, including labeled and unlabled particles can be used insimilar fashion.

[0097] To determine the velocity of a cell, for example, the cellsuspension is injected into main injection well 110 and transportedthrough the channel system by any of a variety of methods, such aspressure differential methods, e.g., applied pressure, wicking,hydrostatic pressure or the like, or electrokinetic methods, both withand without downstream modulation as discussed above.

[0098] The cell is then detected in a detection window as describedabove. As the cell flows through the detection window, signal due tofluorescence, for example, is detected and measured. The signal has anamplitude and duration which are measured, for example, by a computeroperably linked to the detector. The amplitude of the signal correlatesto the number of cells in the window at the time of detection. Theduration of the signal corresponds to how long the cell was in thewindow and thus the velocity is determined by how long the cell took totraverse the detection window. Single cells traversing the detectionwindow are preferred for use in measuring velocity.

[0099] After this determination, one can determine the incubation timeof the cell with a test reagent. With a known velocity, the time thecell spent in contact with a reagent is optionally determined from thetime if injection or mixing of the cell with the reagent in thechannels. Then using the methods of modulation as described above, thisincubation time can be adjusted to suit the particular needs of thesystem being studied. For example, increasing downstream pressure, e.g.,via electrokinetic injection, slows upstream velocity and therebyresults in a longer incubation time, while decreasing downstreampressure, e.g., by electrokinetic withdrawal, increases upstreamvelocity and thereby results in a shorter incubation time.

[0100] VI. Example Bioassays Which Can Be Adapted to the Devices of theInvention

[0101] The present invention provides novel microlaboratory systems andmethods that are useful for performing high-throughput screening assays.In particular, the present invention provides microfluidic devices andmethods of using such devices for screening large numbers of differentcompounds for their effects on a variety of chemical and biochemicalsystems. Methods of controlling, modulating and/or determining the flowrate in these systems are also provided.

[0102] As used herein, the phrase “biochemical system” generally refersto a chemical interaction that involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signalling and other reactions. Further,biochemical systems, as defined herein, also include model systems whichare mimetic of a particular biochemical interaction. Examples ofbiochemical systems of particular interest in practicing the presentinvention include, e.g., receptor-ligand interactions, enzyme-substrateinteractions, cellular signaling pathways, transport reactions involvingmodel barrier systems (e.g., cells or membrane fractions) forbioavailability screening, and a variety of other general systems.Cellular or organismal viability or activity may also be screened usingthe methods and apparatuses of the present invention, e.g., intoxicology studies. Biological materials which are assayed include, butare not limited to, cells, cellular fractions (membranes, cytosolpreparations, mitochondria, nuclei, etc.), agonists and antagonists ofcell membrane receptors (e.g., cell receptor-ligand interactions such ase.g., transferrin, c-kit, viral receptor ligands (e.g., CD4-HIV),cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigottand Power (1993) The Adhesion Molecule FactsBook Academic Press New Yorkand Hulme (ed) Receptor Ligand Interactions A Practical ApproachRickwood and Hames (series editors) IRL Press at Oxford Press N.Y.),toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids,etc.), intracellular receptors (e.g. which mediate the effects ofvarious small ligands, including steroids, thyroid hormone, retinoidsand vitamin D; for reviews see, e.g. Evans (1988) Science, 240:889-895;Ham and Parker (1989) Curr. Opin. Cell Biol., 1:503-511; Burnstein etal. (1989), Ann. Rev. Physiol., 51:683-699; Truss and Beato (1993)Endocr. Rev., 14:459-479), peptides, retro-inverso peptides, polymers ofα-, or β-amino acids (D- or L-), enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, nucleic acids (both linear and cyclic polymerconfigurations), oligosaccharides, proteins, phospho-lipids andantibodies. Synthetic polymers such as hetero-polymers in which a knowndrug is covalently bound to any of the above, such as poly-urethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, and polyacetates arealso assayed. Other polymers are also assayed using the systemsdescribed herein, as would be apparent to one of skill upon review ofthis disclosure. One of skill will be generally familiar with thebiological literature. For a general introduction to biological systems,see, Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, N.Y., (Sambrook); Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (through 1997Supplement) (Ausubel); Watson et al (1987) Molecular Biology of theGene, Fourth Edition The Benjamin/Cummings Publishing Co., Menlo Park,Calif.; Watson et al (1992) Recombinant DNA Second Edition ScientificAmerican Books, N.Y.; Alberts et al. (1989) Molecular Biology of theCell Second Edition Garland Publishing, N.Y.; Pattison (1994) Principlesand Practice of Clinical Virology; Darnell et al., (1990) Molecular CellBiology second edition, Scientific American Books, W.H. Freeman andCompany; Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &Co., Rahway, N.J.; Harrison's Principles of Internal Medicine,Thirteenth Edition, Isselbacher et al. (eds). (1994) Lewin Genes, 5thEd., Oxford University Press (1994); The “Practical Approach” Series ofBooks (Rickwood and Hames (series eds.) by IRL Press at OxfordUniversity Press, N.Y.; The “FactsBook Series” of books from AcademicPress, N.Y., ; Product information from manufacturers of biologicalreagents and experimental equipment also provide information useful inassaying biological systems. Such manufacturers include, e.g., the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

[0103] In order to provide methods and devices for screening compoundsfor effects on biochemical systems, the present invention generallyincorporates models of in vitro systems which mimic a given biochemicalsystem in vivo for which effector compounds are desired. To provided asystem that mimics a biochemical system, it is often useful to have acontrolled flow rate to modulate, for example, a cell incubation period.Additionally, the ability to measure the velocity of the components inthe system is a great advantage to the ability to modulate and controlthe system. These methods are provided in the present invention. Therange of systems against which compounds can be screened and for whicheffector compounds are desired, is extensive. For example, compounds areoptionally screened for effects in blocking, slowing or otherwiseinhibiting key events associated with biochemical systems whose effectis undesirable. For example, test compounds are optionally screened fortheir ability to block systems that are responsible, at least in part,for the onset of disease or for the occurrence of particular symptoms ofdiseases, including, e.g., hereditary diseases, cancer, bacterial orviral infections and the like. To perform screening for large numbers oftest compounds, a microfluidic device that provides continuous flow asthose of the invention, is a great advantage because the testing can becarried out non-stop and with no added time for flow rate adjustments.The invention also provides method of monitoring the flow rates so thatthe adjustments can be quickly and easily determined and made. Compoundswhich slow promising results in these screening assay methods can thenbe subjected to further testing to identify effective pharmacologicalagents for the treatment of disease or symptoms of a disease.

[0104] Alternatively, compounds can be screened for their ability tostimulate, enhance or otherwise induce biochemical systems whosefunction is believed to be desirable, e.g., to remedy existingdeficiencies in a patent.

[0105] Once a model system is selected, batteries of test compounds canthen be applied against these model systems. By identifying those testcompounds that have an effect on the particular biochemical system, invitro, one can identify potential effectors of that system, in vivo.

[0106] In their simplest forms, the biochemical system models employedin the methods and apparatuses of the present invention will screen foran effect of a test compound on an interaction between two components ofa biochemical system, e.g., receptor-ligand interaction,enzyme-substrate interaction, and the like. In this form, thebiochemical system model will typically include the two normallyinteracting components of the system for which an effector is sought,e.g., the receptor and its ligand or the enzyme and its substrate. Withthe methods provided herein, the length of time of the interaction isconveniently determined and modulated if necessary.

[0107] Determining whether a test compound has an effect on thisinteraction then involves contacting the system with the test compoundand assaying for the functioning of the system, e.g., receptor-ligandbinding or substrate turnover. The assayed function is then compared toa control, e.g., the same reaction in the absence of the test compoundor in the presence of a known effector. Typically, such assays involvethe measurement of a parameter of the biochemical system. By “parameterof the biochemical system” is meant some measurable evidence of thesystem's functioning, e.g., the presence or absence of a labeled groupor a change in molecular weight (e.g., in binding reactions, transportscreens), the presence or absence of a reaction product or substrate (insubstrate turnover measurements), or an alteration in electrophoreticmobility (typically detected by a change in elution time of a labeledcompound).

[0108] Although described in terms of two-component biochemical systems,the methods and apparatuses may also be used to screen for effectors ofmuch more complex systems, where the result or end product of the systemis known and assayable at some level, e.g., enzymatic pathways, cellsignaling pathways and the like. Alternatively, the methods andapparatuses described herein are optionally used to screen for compoundsthat interact with a single component of a biochemical system, e.g.,compounds that specifically bind to a particular biochemical compound,e.g., a receptor, ligand, enzyme, nucleic acid, structuralmacromolecule, etc.

[0109] Biochemical system models may also be embodied in whole cellsystems. For example, where one is seeking to screen test compounds foran effect on a cellular response, whole cells are optionally utilized.Modified cell systems may also be employed in the screening systemsencompassed herein. For example, chimeric reporter systems areoptionally employed as indicators of an effect of a test compound on aparticular biochemical system. Chimeric reporter systems typicallyincorporate a heterogenous reporter system integrated into a signalingpathway which signals the binding of a receptor to its ligand. Forexample, a receptor is fused to a heterologous protein, e.g., an enzymewhose activity is readily assayable. Activation of the receptor byligand binding then activates the heterologous protein which then allowsfor detection. Thus, the surrogate reporter system produces an event orsignal which is readily detectable, thereby providing an assay forreceptor/ligand binding. Examples of such chimeric reporter systems havebeen previously described in the art.

[0110] Additionally, where one is screening for bioavailability, e.g.,transport, biological barriers are optionally included. The term“biological barriers” generally refers to cellular or membranous layerswithin biological systems, or synthetic models thereof. Examples of suchbiological barriers include the epithelial and endothelial layers, e.g.vascular endothelia and the like.

[0111] Biological responses are often triggered and/or controlled by thebinding of a receptor to its ligand. For example, interaction of growthfactors, i.e., EGF, FGF, PDGF, etc., with their receptors stimulates awide variety of biological responses including, e.g., cell proliferationand differentiation, activation of mediating enzymes, stimulation ofmessenger turnover, alterations in ion fluxes, activation of enzymes,changes in cell shape and the alteration in genetic expression levels.Another example is the G-protein coupled receptor class of receptorsthat are triggered by a wide variety of peptide and small moleculeagonists, activating the Gα and Gβγ G-protein subunits that havenumerous cellular effects controlled through signal transductionpathways and second messenger modulation. Accordingly, control of theinteraction of the receptor and its ligand may offer control of thebiological responses caused by that interaction.

[0112] Accordingly, in one aspect, the present invention will be usefulin screening for compounds that affect an interaction between a receptormolecule and its ligands. As used herein, the term “receptor” generallyrefers to one member of a pair of compounds which specifically recognizeand bind to each other. The other member of the pair is termed a“ligand.” Thus, a receptor/ligand pair may include a typical proteinreceptor, usually membrane associated, and its natural ligand, e.g.,another protein or small molecule. Receptor/ligand pairs may alsoinclude antibody/antigen binding pairs, complementary nucleic acids,nucleic acid associating proteins and their nucleic acid ligands. Alarge number of specifically associating biochemical compounds are wellknown in the art and can be utilized in practicing the presentinvention.

[0113] Traditionally, methods for screening for effectors of areceptor/ligand interaction have involved incubating a receptor/ligandbinding pair in the presence of a test compound. The level of binding ofthe receptor/ligand pair is then compared to negative and/or positivecontrols. Where a decrease in normal binding is seen, the test compoundis determined to be an inhibitor of the receptor/ligand binding. Wherean increase in that binding is seen, the test compound is determined tobe an enhancer or inducer of the interaction.

[0114] A similar, and perhaps overlapping, set of biochemical systemsincludes the interactions between enzymes and their substrates. The term“enzyme” as used herein, generally refers to a protein which acts as acatalyst to induce a chemical change in other compounds or “substrates.”

[0115] Typically, effectors of an enzyme's activity toward its substrateare screened by contacting the enzyme with a substrate in the presenceand absence of the compound to be screened and under conditions optimalfor detecting changes in the enzyme's activity. After a set time forreaction, the mixture is assayed for the presence of reaction productsor a decrease in the amount of substrate. The amount of substrate thathas been catalyzed is them compared to a control, i.e., enzyme contactedwith substrate in the absence of test compound or presence of a knowneffector. As above, a compound that reduces the enzymes activity towardits substrate is termed an “inhibitor,” whereas a compound thataccentuates that activity is termed an “inducer.”

[0116] Generally, the various screening methods encompassed by thepresent invention involve the serial introduction of a plurality of testcompounds into a microfluidic device. Once injected into the device, thetest compound is screened for effect on a biological system using acontinuous serial or parallel assay orientation.

[0117] As used herein, the term “test compound” refers to the collectionof compounds that are to be screened for their ability to affect aparticular biochemical system. Test compounds may include a wide varietyof different compounds, including chemical compounds, mixtures ofchemical compounds, e.g., polysaccharides, small organic or inorganicmolecules, biological macromolecules, e.g., peptides, proteins, nucleicacids, or an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues, naturally occurring orsynthetic compositions. Depending upon the particular embodiment beingpracticed, the test compounds are provided, e.g., injected into amicrofluidic device, free in solution, or are optionally attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports are employed for immobilization of the test compounds. Examplesof suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films, glass beads,polyaminemethylvinylether maleic acid copolymer, amino acid copolymer,ethylene-maleic acid copolymer, nylon, silk, etc. Additionally, for themethods and apparatuses described herein, test compounds are screenedindividually, or in groups. Group screening is particularly useful wherehit rates for effective test compounds are expected to be low such thatone would not expect more than one positive result for a given group.Alternatively, such group screening is used where the effects ofdifferent test compounds are differentially detected in a single system,e.g., through electrophoretic separation of the effects, or differentiallabeling which enables separate detection.

[0118] Test compounds are commercially available, or derived from any ofa variety of biological sources apparent to one of skill and asdescribed, supra. In one aspect, a tissue homogenate or blood samplefrom a patient is tested in the assay systems of the invention. Forexample, in one aspect, blood is tested for the presence or activity ofa biologically relevant molecule. For example, the presence and activitylevel of an enzyme are detected by supplying and enzyme substrate to thebiological sample and detecting the formation of a product using anassay systems of the invention. Similarly, the presence of infectiouspathogens (viruses, bacteria, fungi, or the like) or cancerous tumorscan be tested by monitoring binding of a labeled ligand to the pathogenor tumor cells, or a component of the pathogen or tumor such as aprotein, cell membrane, cell extract or the like, or alternatively, bymonitoring the presence of an antibody against the pathogen or tumor inthe patient's blood. For example, the binding of an antibody from apatient's blood to a viral protein such as an HIV protein is a commontest for monitoring patient exposure to the virus. Many assays fordetecting pathogen infection are well known, and are adapted to theassay systems of the present invention.

[0119] Biological samples are derived from patients using well knowntechniques such as venipuncture or tissue biopsy. Where the biologicalmaterial is derived from non-human animals, such as commerciallyrelevant livestock, blood and tissue samples are conveniently obtainedfrom livestock processing plants. Similarly, plant material used in theassays of the invention are conveniently derived from agricultural orhorticultural sources. Alternatively, a biological sample can be from acell or blood bank where tissue and/or blood are stored, or from an invitro source such as a culture of cells. Techniques and methods forestablishing a culture of cells for use as a source for biologicalmaterials are well known to those of skill in the art. Freshney Cultureof Animal Cells a Manual of Basic Technique, Third Edition Wiley-Liss,New York (1994) provides a general introduction to cell culture.

[0120] Any of the above assays or screens are optionally performed inthe systems of the invention. When a particular flow rate is desired,the methods of the invention are used to modulate the downstreampressure to provide a particular flow rate. The velocity of thematerials in the assay is optionally measured by the methods of thepresent invention. The velocity and/or incubation time of a cell is thenoptionally controlled or modulated by the techniques described above,such as by use of a wick or electrokinetic injection. Using thesemethods, the assays are optionally run continuously and consistently atdesired flow rates.

[0121] VII. Description of Microfluidic Devices and Systems

[0122] The wick and electrokinetic injections of the invention aretypically used to run bioassays of the type described above in themicrofluidic devices and systems described below. To provide continuousand consistent flow in the bioassays, the microfluidic devices below areoptionally fitted with an absorbent material fabricated into a well,such as Porex™ plug fitted into a waste well of the device, or a well isprovided for later insertion of a wick or other absorbent material, suchas piece of paper. The absorbent material is provided to modulate orcontrol the flow rate of materials within the device.

[0123] As used herein, the term “microscale” or “microfabricated”generally refers to structural elements or features of a device whichhave at least one fabricated dimension in the range of from about 0.1μto about 500μ. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or feature, suchas a channel, well, or absorbent wick, having such a dimension. Whenused to describe a fluidic element, such as a passage, chamber orconduit, the terms “microscale,” “microfabricated” or “microfluidic”generally refer to one or more fluid passages, chambers or conduitswhich have at least one internal cross-sectional dimension, e.g., depth,width, length, diameter, etc., that is less than 500μ, and typicallybetween about 0.1μ and about 500μ In the devices of the presentinvention, the microscale channels or chambers preferably have at leastone cross-sectional dimension between about 0.1μ and 200μ, morepreferably between about 0.1μ and 100μ, and often between about 0.1μ and20μ. Accordingly, the microfluidic devices or systems prepared inaccordance with the present invention typically include at least onemicroscale channel, usually at least two intersecting microscalechannels, and often, three or more intersecting channels disposed withina single body structure. Channel intersections may exist in a number offormats, including cross intersections, “T” intersections, or any numberof other structures whereby two channels are in fluid communication.

[0124] Typically, the microfluidic devices described herein willcomprise a top portion, a bottom portion, and an interior portion,wherein the interior portion substantially defines the channels andchambers of the device.

[0125]FIG. 3 illustrates a body structure 301, for a microfluidicdevice. In preferred aspects, the bottom portion of the device comprisesa solid substrate that is substantially planar in structure, and whichhas at least one substantially flat upper surface 305. A variety ofsubstrate materials are optionally employed as the bottom portion.Typically, because the devices are microfabricated, substrate materialswill be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, printing, and other techniques. The substrate materials arealso generally selected for their compatibility with the full range ofconditions to which the microfluidic devices are exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents.

[0126] In additional preferred aspects, the substrate materials willcomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, ABS(acrylonitrile-butadiene-styrene copolymer), and the like. Suchpolymeric substrates are readily manufactured using availablemicrofabrication techniques, as described above, or from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing or stamping, or by polymerizing the polymeric precursormaterial within the mold (See, U.S. Pat. No. 5,512,131). Such polymericsubstrate materials are preferred for their ease of manufacture, lowcost and disposability, as well as their general inertness to mostextreme reaction conditions. Again, these polymeric materials mayinclude treated surfaces, e.g., derivatized or coated surfaces, toenhance their utility in the microfluidic systems, e.g., provideenhanced fluid direction, e.g., as described in published PCTapplication, WO 98/05424, and which is incorporated herein by referencein its entirety for all purposes.

[0127] The channels and/or chambers or wells of the microfluidic devicesare typically fabricated into the upper surface of the bottom substrate,as microscale grooves or indentations, such as first channel region 340,using the above described microfabrication techniques. In themicrofluidic devices prepared in accordance with the methods describedherein, the top portion also includes a plurality of apertures, such asholes or ports 310, 315, and 320 disposed therethrough.

[0128] The first planar surface of the top substrate is then mated,e.g., placed into contact with, and bonded to the planar surface of thebottom substrate, covering and sealing the grooves and/or indentationsin the surface of the bottom substrate, to form the channels and/orchambers (i.e., the interior portion) of the device at the interface ofthese two components. The holes or wells in the top portion of thedevice are oriented such that they are in communication with at leastone of the channels and/or chambers formed in the interior portion ofthe device from the grooves or indentations in the bottom substrate. Inthe completed device, these holes function as reservoirs or wells forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes are optionally placed into contact with fluidswithin the device, allowing application of electric fields andelectrokinetic injection or withdrawal of buffer along the channels ofthe device to control and direct fluid transport within the device. Inanother embodiment, the holes or wells contain wicks or other absorbentmaterials to provide for flow and modulation, e.g., for continuous ordiscontinuous flow applications.

[0129] In many embodiments, the microfluidic devices will include anoptical detection window disposed across one or more channels and/orchambers of the device. Optical detection windows are typicallytransparent such that they are capable of transmitting an optical signalfrom the channel/chamber over which they are disposed. Optical detectionwindows may merely be a region of a transparent cover layer, e.g., wherethe cover layer is glass or quartz, or a transparent polymer material,e.g., PMMA, polycarbonate, etc. Alternatively, where opaque substratesare used in manufacturing the devices, transparent detection windowsfabricated from the above materials are separately manufactured into thedevice.

[0130] These devices are used in a variety of applications, including,e.g., the performance of high throughput screening assays in drugdiscovery, immunoassays, diagnostics, genetic analysis, call analysisand the like. As such, the devices described herein, will often includemultiple sample introduction ports or reservoirs, for the parallel orserial introduction and analysis of multiple samples. Alternatively,these devices are coupled to a sample introduction port, e.g., apipetor, which serially introduces multiple samples into the device foranalysis. Examples of such sample introduction systems are described ine.g., U.S. patent application Ser. Nos. 08/761,575 and 08/760,446(Attorney Docket Nos. 17646-000410 and 17646-000510, respectively) eachof which was filed on Dec. 6, 1996, and is hereby incorporated byreference in its entirety for all purposes.

[0131] The wells and/or injection ports described above are used in thepresent invention for electrokinetic injection or withdrawal orplacement of a wick or other absorbent materials. The electrokineticinjection or withdrawal and/or wick is then optionally used to controlthe flow rates through the channels of the device as described above.

[0132] One example is schematically illustrated in FIG. 3A as described.FIGS. 3B and 3C schematically illustrate alternate configurations of theinterface between the wick and the microfluidic device. For example,FIG. 3B shows a partial view of the body structure 301 of a microfluidicdevice that includes a well 310 and a channel 352 fluidly connected tothe well 310. A wick 354 is placed into well 310 substantially asdescribed above. In preferred aspects, the wick is a cylindrical bundleof absorbent material, e.g., as described herein, and resembles acigarette filter in its structure and consistency. The cylindrical shapeof the wick allows the wick to be fittedly inserted into a circularwell, e.g., well 310, although the shape of the wick can be adjusted toaccommodate any well shape. Typically, such cylindrical wicks comprisebundles of fibrous materials which are absorbent in their own right,and/or are absorbent by virtue of their being bundled together as aporous structure, giving rise to capillary-type wicking.

[0133] As shown in FIG. 3B, the wick is illustrated as extending wellabove the upper edge 305 of well 310, to provide a sufficient amount ofmaterial to prevent saturation of the wick. Of course, in someinstances, a saturated wick may be desirable, e.g., where extremely slowwicking or flow rates are desired. In such cases, wicking from the well310 will be limited in large part by the rate of evaporation from thewick 354. However, because evaporation rates vary with the relativeambient humidity, it is often desirable for wicking to benon-evaporation limited. Again, this is accomplished in a first aspectby providing a sufficient amount of wicking material. Because fluidvolumes in the microfluidic devices of the invention are extremelysmall, e.g., on the order of microliters deposited in the wells, a wickthat merely fills the volume of the well, or is slightly larger, isgenerally sufficient. Further, larger wicks, with their greater surfaceareas, typically evaporate greater amounts of fluid, thus reducing thechance that they will reach saturation during the operation to which thedevice is being put, and thereby preventing evaporation from becomingthe rate limiting event.

[0134] An alternate wick structure is illustrated in FIG. 3C. As shown,the wick 354 again comprises a cylindrical bundle of absorbent materialthat is inserted into well 310. However, as shown, the wick 354 includesa cap 358 that substantially seals well 310, to prevent excessevaporation of material in the well, which evaporation can result infaster flow of material from channel 352 into well 310, and in somecases, a faster wicking rate, thereby resulting in a variable flow ratedepending upon ambient humidity. In the case of the device shown in FIG.3C, cap 358 substantially seals well 310 by extending beyond the edge ofthe wick 354 and overlapping the upper edges 305 of the well 310. Inorder to prevent back-pressure within the well 310 from slowing orstopping flow, cap 358 includes a vent or hole 360 disposed through itto maintain the well substantially at ambient pressure.

[0135] A. Device Integration

[0136] Although the devices and systems specifically illustrated hereinare generally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example, inthe present invention, the devices and systems described include methodsfor detection and monitoring of the materials that are used for thedetermination of velocity. Since the present invention uses downstreampressure to control the flow rates of materials in the system,downstream devices to control the pressure are included in the system.For example, the devices and systems described will optionally includestructures, reagents and systems for performing virtually any number ofoperations both upstream and downstream from the operations specificallydescribed herein.

[0137] Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification,amplification, cellular activation, labeling reactions, dilution,aliquoting, and the like.

[0138] Similarly, downstream operations may include similar operations,including, e.g., separation of sample components, labeling ofcomponents, assays and detection operations, electrokinetic injection orwithdrawal and wicking. Assay and detection operations include, withoutlimitation, probe interrogation assays, e.g., nucleic acid hybridizationassays utilizing individual probes, free or tethered within the channelsor chambers of the device and/or probe arrays having large numbers ofdifferent, discretely positioned probes, receptor/ligand assays,immunoassays, and the like.

[0139] B. Instrumentation

[0140] In the present invention, the materials in the channels aremonitored and detected so that velocity may be determined. From velocitymeasurements, decisions are then made regarding flow control mechanisms.Various flow rate control methods, such as a wick or an electrokineticor pressure based downstream an injection, are then used to controland/or change the measured velocity. Sample incubation times (e.g., forcell samples) are also measured and altered with the above methods.Additional available instrumentation may be used to obtain and analyzethese measurements.

[0141] The systems described herein generally include microfluidicdevices, as described above, in conjunction with additionalinstrumentation for controlling fluid transport, flow rate and directionwithin the devices, detection instrumentation for detecting or sensingresults of the operations performed by the system, processors, e.g.,computers, for instructing the controlling instrumentation in accordancewith preprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretation in a readily accessiblereporting format.

[0142] C. Controller

[0143] A variety of controlling instrumentation is optionally utilizedin conjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluids and/or materialswithin the devices of the present invention. For example, in many cases,fluid transport and direction are controlled in whole or in part, usingpressure based flow systems that incorporate external or internalpressure sources to drive fluid flow. Internal sources includemicrofabricated pumps, e.g., diaphragm pumps, thermal pumps, lamb wavepumps and the like that have been described in the art. See, e.g. U.S.Pat. Nos. 5,271,724, 5,277,556, and 5,375,979 and Published PCTApplication Nos. WO 94/05414 and WO 97/02357. In such systems, fluiddirection is often accomplished through the incorporation ofmicrofabricated valves, which restrict fluid flow in a controllablemanner. See, e.g., U.S. Pat. No. 5,171,132.

[0144] As noted above, the systems described herein can utilizeelectrokinetic material direction and transport systems. As such, thecontroller systems for use in conjunction with the microfluidic devicestypically include an electrical power supply and circuitry forconcurrently delivering appropriate voltages to a plurality ofelectrodes that are placed in electrical contact with the fluidscontained within the microfluidic devices. Examples of particularlypreferred electrical controllers include those described in, e.g.,published PCT application WO 98/05424, the disclosure of which is herebyincorporated herein by reference in its entirety for all purposes. Inbrief, the controller uses electric current control in the microfluidicsystem. The electrical current flow at a given electrode is directlyrelated to the ionic flow along the channel(s) connecting the reservoirin which the electrode is placed. This is in contrast to the requirementof determining voltages at various nodes along the channel in a voltagecontrol system. Thus the voltages at the electrodes of the microfluidicsystem are set responsive to the electric currents flowing through thevarious electrodes of the system. This current control is lesssusceptible to dimensional variations in the process of creating themicrofluidic system in the device itself. Current control permits fareasier operations for pumping, valving, dispensing, mixing andconcentrating subject materials and buffer fluids in a complexmicrofluidic system. Current control is also preferred for moderatingundesired temperature effects within the channels.

[0145] Typically, the controller systems are appropriately configured toreceive a microfluidic device as described herein. In particular, thecontroller and/or detector (as described in greater detail, below),includes a stage upon which the device of the invention is mounted tofacilitate appropriate interfacing between the controller and/ordetector and the device. Typically, the stage includes an appropriatemounting/alignment structural element, such as a nesting well, alignmentpins and/or holes, asymmetric edge structures (to facilitate properdevice alignment), and the like.

[0146] The controlling instrumentation discussed above is also used toprovide for electrokinetic injection or withdrawal of materialdownstream of the region of interest to control an upstream flow rate.The same instrumentation and techniques described above are alsoutilized to inject a fluid into a downstream port to function as a flowcontrol element.

[0147] D. Detector

[0148] In the microfluidic systems described herein, a variety ofdetection methods and systems are employed, depending upon the specificoperation that is being performed by the system. Often, a microfluidicsystem will employ multiple different detection systems for monitoringthe output of the system. Detection systems of the present invention areused to detect and monitor the materials in the detection window. Oncedetected, the flow rate and velocity of particles in the channels isoptionally measured and controlled as described above.

[0149] Examples of detection systems include optical sensors,temperature sensors, pressure sensors, pH sensors, conductivity sensors,and the like. Each of these types of sensors is readily incorporatedinto the microfluidic systems described herein. In these systems, suchdetectors are placed either within or adjacent to the microfluidicdevice or one or more channels, chambers or conduits of the device, suchthat the detector is within sensory communication with the device,channel, or chamber. The phrase “within sensory communication” of aparticular region or element, as used herein, generally refers to theplacement of the detector in a position such that the detector iscapable of detecting the property of the microfluidic device, a portionof the microfluidic device, or the contents of a portion of themicrofluidic device, for which that detector was intended. For example,a pH sensor placed in sensory communication with a microscale channel iscapable of determining the pH of a fluid disposed in that channel.Similarly, a temperature sensor placed in sensory communication with thebody of a microfluidic device is capable of determining the temperatureof the device itself.

[0150] Particularly preferred detection systems include opticaldetection systems for detecting an optical property of a material withinthe channels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. Suchoptical detection systems are typically placed adjacent to a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the materials spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material. As such, the detectionsystem will typically include collection optics for gathering a lightbased signal transmitted through the detection window, and transmittingthat signal to an appropriate light detector. Microscope objectives ofvarying power, field diameter, and focal length are readily utilized asat least a portion of this optical train. The light detectors areoptionally photodiodes, avalanche photodiodes, photomultiplier tubes,diode arrays, or in some cases, imaging systems, such as charged coupleddevices (CCDs) and the like. In preferred aspects, photodiodes areutilized, at least in part, as the light detectors. The detection systemis typically coupled to a computer (described in greater detail below),via an analog to digital or digital to analog converter, fortransmitting detected light data to the computer for analysis, storageand data manipulation.

[0151] In the case of fluorescent materials, the detector will typicallyinclude a light source which produces light at an appropriate wavelengthfor activating the fluorescent material, as well as optics for directingthe light source through the detection window to the material containedin the channel or chamber. The light source any number of light sourcesthat provides the appropriate wavelength, including lasers, laser diodesand LEDs. Other light sources required for other detection systems. Forexample, broad band light sources are typically used in lightscattering/transmissivity detection schemes, and the like. Typically,light selection parameters are well known to those of skill in the art.

[0152] The detector may exist as a separate unit, but is preferablyintegrated with the controller system, into a single instrument.Integration of these functions into a single unit facilitates connectionof these instruments with the computer (described below), by permittingthe use of few or a single communication port(s) for transmittinginformation between the controller, the detector and the computer.

[0153] E. Computer

[0154] As noted above, either or both of the controller system and/orthe detection system are coupled to an appropriately programmedprocessor or computer which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. As such, thecomputer is typically appropriately coupled to one or both of theseinstruments (e.g., including an analog to digital or digital to analogconverter as needed).

[0155] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation. The computerthen receives the data from the one or more sensors/detectors includedwithin the system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like.

[0156] In the present invention, the computer typically includessoftware for the monitoring of materials in the channels, so that flowrate and velocity may be determined. Additionally the software isoptionally used to control electrokinetic injection or withdrawal ofmaterial. The electrokinetic or withdrawal is used to modulate the flowrate as described above.

[0157] F. Kits

[0158] Generally, the microfluidic devices described herein are packagedto include many if not all of the necessary reagents for performing thedevice's preferred function. Such kits also typically includeappropriate instructions for using the devices and reagents, and incases where reagents are not predisposed in the devices themselves, withappropriate instructions for introducing the reagents into the channelsand/or chambers of the device. In this latter case, these kitsoptionally include special ancillary devices for filling themicrofluidic channels, e.g., appropriately configured syringes/pumps, orthe like. In the former case, such kits typically include a microfluidicdevice with necessary reagents predisposed in the channels/chambers ofthe device. Generally, such reagents are provided in a stabilized form,so as to prevent degradation or other loss during prolonged storage,e.g., from leakage. A number of stabilizing processes are widely usedfor reagents that are to be stored, such as the inclusion of chemicalstabilizers (i.e., enzymatic inhibitors, microcides/bacteriostats,anticoagulants), the physical stabilization of the material, e.g.,through immobilization on a solid support, entrapment in a matrix (i.e.,a gel), lyophilization, or the like.

[0159] Such kits also optionally include an absorbent material that isoptionally used as a wick to sustain flow rates as described above.Additionally the kits may come with the wick or absorbent materialpredisposed in the devices to modulate and/or sustain flow rates.Accordingly, one feature of the invention is the manufacture ofmicrofluidic devices comprising absorbent materials, such as any ofthese described herein.

[0160] The discussion above is generally applicable to the aspects andembodiments of the invention described above.

[0161] Moreover, modifications can be made to the method and apparatusdescribed herein without departing from the spirit and scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses including the following.

[0162] The use of a microfluidic system containing at least a firstsubstrate and having a first channel and a second channel intersectingthe first channel, at least one of the channels having at least onecross-sectional dimension in a range from 0.1 to 500 μm, in order totest the effect of each of a plurality of test compounds on abiochemical system. The system including a wick or other absorbentmaterial.

[0163] The use of a microfluidic system as described herein, wherein abiochemical system flows through one of said channels substantiallycontinuously, providing for, e.g., sequential testing of said pluralityof test compounds.

[0164] The use of an absorbent material in a microfluidic device asdescribed herein to modulate or achieve flow in the channels.

[0165] The use of an electrokinetic injection in a microfluidic deviceas described herein to modulate or achieve flow in the channels.

[0166] The use of a combination of wicks, electrokinetic injection andpressure based flow elements in a microfluidic device as describedherein to modulate or achieve continuous flow in the channels.

[0167] An assay utilizing a use of any one of the microfluidic systemsor substrates described herein.

[0168] Microfluidic devices and bioassays which can be adapted to thepresent invention include various PCT applications and issued U.S.Patents, such as, U.S. Pat. Nos. 5,699,157 (J. Wallace Parce) issuedDec. 16, 1997, 5,779,868 (J. Wallace Parce et al.) issued Jul. 14, 1998,5,800,690 (Calvin Y. H. Chow et al.) issued Sep. 1, 1998, and 5,842,787(Anne R. Kopf-Sill et al.) issued Dec. 1, 1998; and published PCTapplications, such as, WO 98/00231, WO 98/00705, WO 98/00707, WO98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO 98/45929, WO98/46438, and WO 98/49548, which are all incorporated herein byreference.

EXAMPLES

[0169] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar or desirably different results.

Example 1 Control of Flow Rate Using Wicking

[0170] Live cells labeled with a fluorescent DNA dye were loaded into adevice as in FIG. 3. Cells were detected as they flowed past fluorescentreader in reading area 345. Each peak represents a cell or multiplecells depending how many were in the read area at once.

[0171] An empty well was designated as waste well 310, and a piece ofKimwipe™ (3 mm wide and 1.5 cm long) was placed in the waste well, as awick, so that one end was touching the bottom of the well, and the restof the piece climbed up and lay flat outside the well on upper surface305 of the device. A 2.5 microliter pipettor with 2 microliters ofliquid in the tip was used to press the wick against the wall of thewell. The 2 microliters of liquid was expelled to help start the wickingaction. The wick was then lifted from the surface of the microfluidicdevice to form a bubble as shown in FIG. 3, to facilitate evaporationfrom the wick to maintain the wicking action.

[0172] In other embodiments, evaporation can be limited or eliminated,e.g., by applying a cap to the absorbent material (see e.g., FIG. 3C). Agraduated wick, e.g., which has gradually increasing width dimensionscan also be used to regulate flow rates.

[0173] Reading was started after the wick was placed in well 310, atabout 400 seconds. The detected signals are shown in FIG. 7. The narrowpeaks indicate a high flow rate because they flowed through the readarea quickly, giving the peak a short duration. FIGS. 8-10-show theeffects after the wick was removed at 500 seconds. The flow rate sloweddown as shown in FIG. 8, indicated by fewer cells crossing the read areaand being detected. In addition, those that were detected were increasedin peak width indicating a slower flow rate. At about 1000 seconds, theflow rate eventually stopped without the wick. FIGS. 11 and 12, show thesignals detected after the wick was replaced in the well at about 1300seconds. With the wick functioning to draw the sample through thechannels, the flow rate resumed and continued at a good rate. At 2700seconds, the flow rate was still good as indicated by the narrow peakwidths in FIG. 12.

[0174] Without a wick to provide continuous flow, the flow ratecontinued for only 500 seconds compared to 1400 seconds (from 1300 to2700) of sustained flow using a wick. Therefore, the wick providedconvenient method to increase flow rates and sustain them in amicrofluidic channel.

Example 2 Control of Flow Rates Using Electrokinetic Injection

[0175] Dye Experiments

[0176] In this experiment, a buffer was injected into side channels of amicrofluidic device to demonstrate the ability of side channelelectrokinetic injection to rapidly control the flow rate in the mainchannel of a microfluidic device. A dye was used in the main channel ofFIG. 2, with dilution of the dye being an indicator of the effect ofside channel buffer injection on the main channel flow rate.

[0177] The dye used was Bodipy-Fluorescein (Molecular Probes) dissolvedin Hank's Balanced Salt Solution which is the isotonic-high salt bufferadded to buffer well 220. The low ionic strength buffer, 30 mM HEPES, pH7.0 in deionized water, was added to buffer well 215. The wick was a 3mm strip of Kimwipe, placed in well 225. 10 μl of each solution wasadded to buffer well 220 and buffer well 215 respectively. Current wasthen applied to side channels 235 and 240, and no current to mainchannel 210 as pictured in FIG. 2. The reading or detecting area was 0.5mm after the buffer injection intersection in main channel region 245.

[0178]FIG. 4 shows the effect of the buffer from a side channel beinginjected into the main channel. As the buffer was injected, it dilutedthe dye in the main channel. This demonstrated the effectiveness of thebuffer pumping from the side channel in the present configuration. FIG.5 shows that toggling the current to the side channels on and offrapidly changes the dilution of the dye in the main channel indicatingrapid control of the flow rate from the side channel.

[0179] Cell Experiments

[0180] After the dye experiment proved the ability of the side channelinjection to modulate the flow rate in the main channel, a cellexperiment was performed to demonstrate the effect of side channelelectrokinetic injection on the velocity of cells in a microfluidicdevice. In this experiment, the detection area was placed upstream ofthe buffer injection site, so that no dilution of the cell suspensionwould occur before detection.

[0181] The cells used were THP-1 cells cultured as recommended by theATCC in RPMI 1640 containing 10% fetal bovine serum, 1 mM pyruvate, 2 mML-glutamate, 50 μM β-mercaptoethanol, 10 mM HEPES. The cells were loadedwith Calcein-AM dye at 1 μM for 15 minutes at room temperature in HBSScontaining 1 mg/ml BSA, pelleted at 300×g for 5 minutes, and resuspendedin Cell Buffer (HBSS containing 1 mg/ml BSA, 20 mM HEPES, 10% w/vOptiprep, specific density adjustment agent). 10 μl of cell suspensionor buffers were added to injection well 205. Low ionic strength bufferwas injected as in the dye experiments; however the reading area was 2mm upstream of the buffer injection, in main channel region 240.

[0182]FIG. 6 shows the effect of buffer injections from the side channelon the flow rate of cells in the main channel. The injection current wastoggled from 2 μA for 10 seconds to 0 for 10 seconds. The width of thepeaks as cells pass in front of the fluorescence detector varied withthe velocity of the cells. The higher the velocity the narrower thepeak. The higher the buffer injection current the slower the cellmovement. When the current was turned off, the cell velocity returned tothe higher rate.

[0183] One of the advantages of this configuration is that the cellvelocities are controlled electronically without moving parts, and theelectro-osmotically pumped buffer composition can be optimized forpumping efficiency without regard to deleterious effects on the cellssince it contacts the cells after the assay measurements are made.

Example 3 Calcium Flux Assay

[0184] Using a continuous flow format in planar chips using a wick togenerate constant pressure driven flow, cells were mixed with an agonistand down-stream fluorescence detectors were used to monitor indicatorcells using Fluo-3 (Molecular Probes Inc.) calcium sensitive fluorescentdye as a probe for receptor activation of calcium fluxes. THP-1 cellswere loaded with Fluo-3 by incubation with a 4 μM concentration of theFluo-3 AM (acetoxymethyl ester) in Hank's Balanced Salt Solution(HBSS)containing 20 mM HEPES, pH 7.0, and 1 mg/ml bovine serum albumin. Aftera 40 minute incubation at 37° C., Syto-6, a fluorescent DNA stain, wasadded to 2.5 μM, and the cells were incubated for an additional 10minutes at room temperature. The cells were then washed free of excessdye by pelleting at 300×g for 5 minutes and resuspending andrepelleting. The cells were resuspended in Cell Buffer (HBSS containing20 mM HEPES, pH 7.0, 1 mg/ml BSA and 10% Optiprep). The THP-1 cells weretested for UTP-activated calcium fluxes mediated through the purinergic,P2Y receptor by adding different concentrations of UTP from 0-3 μM tothe cells in the microfluidic device and detecting the calcium responseusing a blue LED to excite the intracellular Fluo-3 and the Syto-62.

[0185] The results of these tests are displayed in FIG. 13 showing thespikes of fluorescence as the cells pass the detector. The lower traceshows that the fluorescence of the DNA staining dye is not affected bythe UTP treatment, and the upper trace shows that the fluorescence ofthe calcium sensitive dye increases with UTP treatment in a dosedependent manner, indicating an increase in intracellular free calcium.By taking the ratio of the two fluorescent dyes, it is possible tonormalize the calcium flux response in the THP-1 cells since the DNAstaining intensity is a constant in diploid, resting cells, and therebyquantitate the increase in calcium concentration.

[0186] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed:
 1. A method of modulating flow rate of a first materialin a first channel region in a microfluidic system, the methodcomprising: (i) providing a body structure having at least twointersecting channels fabricated therein, wherein said first channelregion is a portion of at least one of the at least two intersectingchannels; and, (ii) modulating pressure downstream of the first channelregion, thereby increasing or decreasing flow rate of said firstmaterial in said first channel region.
 2. The method of claim 1, whereinthe microfluidic system further comprises at least one well fluidlycoupled to the first channel region, wherein the pressure is modulatedby placing an absorbent material in the at least one well, whichabsorbent material is fluidly coupled to the first material.
 3. Themethod of claim 2, wherein the pressure is modulated by placing theabsorbent material at a junction between at least one of the at leasttwo intersecting channels and the at least one well, whereby theabsorbent material absorbs fluid in the well, thereby modulating flow ofthe fluid into the well and regulating the flow rate of said firstmaterial.
 4. The method of claim 2, wherein the at least one well is awaste well in fluidic communication with the first channel region; and,wherein the pressure is modulated by placing an absorbent material inthe waste well, which absorbent material is positioned proximal to ajunction between the waste well and a second channel region, whichsecond channel region is fluidly coupled to the first channel region,whereby the absorbent material absorbs fluid in the waste well, therebymodulating flow of the fluid into the waste well and regulating the flowrate of said first material.
 5. The method of claim 2, wherein theabsorbent material is selected from: a solid material, a porousmaterial, a gel, a polymer, a high salt fluid, a thermoplastic polymer,a porous plastic, and a polyolefin resin.
 6. The method of claim 2,wherein the material is selected from: paper, dried polyacrylamide, drysephadex, a dextran particle, a polyethylene particle, a polypropyleneparticle, a high molecular weight polyethylene particle, apolyvinylidene fluoride particle, an ethylene-vinyl acetate particle, apolytetrafluoroethylene particle, a stryene-acrylonitrile particle, apolysulfone particle, a polycarbonate particle, a polyhthalate particle,and combinations thereof.
 7. The method of claim 2, wherein the materialfurther comprises a surfactant.
 8. The method of claim 2, wherein the atleast one well has an upper edge, and wherein the absorbent materialprojects beyond the upper edge.
 9. The method of claim 2, wherein theabsorbent material is positioned entirely within the at least one well.10. The method of claim 1, wherein the flow rate is controlled byelectrokinetic injection or withdrawal of a second material downstreamof the first channel region, wherein the electrokinetic injection of thesecond material modulates pressure downstream of the first material,thereby modulating the flow rate of the first material.
 11. The methodof claim 1, wherein the microfluidic system further comprises at leastone well fluidly coupled to the first channel region, wherein thepressure is modulated by placing an absorbent material in the at leastone well, which absorbent material is fluidly coupled to the firstmaterial, wherein the absorbent material is pre-wetted prior toplacement in the at least one well.
 12. The method of claim 1, whereinthe microfluidic system further comprises at least one well fluidlycoupled to the first channel region, wherein the pressure is modulatedby placing an absorbent material in the at least one well, whichabsorbent material is fluidly coupled to the first material, wherein theabsorbent material is not pre-wetted prior to placement in the at leastone well.
 13. The method of claim 1, the method further comprising,(iii) electrokinetic injection of a second material downstream of thefirst channel region, thereby modulating the flow rate if the firstmaterial, and (iv) monitoring the flow rate of material in the firstchannel region.
 14. The method of claim 13, wherein the monitoring isperformed before injection, after injection, or before and afterinjection.
 15. The method of claim 13, wherein step (iv) comprises: (v)detecting a signal from the first component for a period of time, thesignal having an amplitude and a duration, and (vi) measuring theduration and amplitude of the signal, thereby monitoring the flow rateof material.
 16. The method of claim 1, wherein the pressure iscontrolled by placing a wick comprising an absorbent material in fluidcommunication with the first channel region.
 17. The method of claim 16,wherein the microfluidic system further comprises at least one well influid communication with the first channel region, wherein the pressureis modulated by placing the absorbent material in the at least one well.18. The method of claim 1, wherein the pressure is controlled by etchinga network of capillaries downstream of the first channel region.
 19. Amethod of modulating flow rate of a sample in a microfluidic system, themethod comprising: (i) providing a body structure having at least onemicrofluidic channel fabricated therein, (ii) flowing the sample throughthe at least one microfluidic channel; and, (ii) increasing ordecreasing pressure downstream of a selected site comprising the samplewithin the channel, thus modulating flow rate of the sample in themicrofluidic channel.
 20. The method of claim 19, wherein the bodystructure further comprises at least two intersecting microfluidicchannels fabricated therein.
 21. The method of claim 19, wherein thebody structure further comprises at least two intersecting microfluidicchannels fabricated therein and said increasing or decreasing pressureis performed by electrokinetic injection or withdrawal of fluid into orfrom the at least two intersecting channels at a site downstream from aselected site comprising the sample.
 22. The method of claim 19, whereinthe increasing or decreasing pressure occurs electrokinetically.
 23. Themethod of claim 19, wherein the microfluidic system further comprises atleast one well fluidly coupled to the at least one microfluidic channel,wherein the flow rate is modulated by placing an absorbent material inthe at least one well, which absorbent material is fluidly coupled tothe sample.
 24. A method of modulating flow rate of a sample in amicrofluidic system, the method comprising: (i) providing a bodystructure having at least one microfluidic channel fabricated therein,(ii) flowing the sample through the at least one microfluidic channel;and, (ii) providing an absorbent material downstream of a selected sitecomprising the sample, thus modulating flow rate of the sample in themicrofluidic channel.
 25. The method of claim 24, wherein the absorbentmaterial comprises a wick.
 26. The method of claim 24, wherein themicrofluidic system further comprises at least one well fluidly coupledto the at least one channel, wherein the flow rate is modulated byplacing an absorbent material in the at least one well, which absorbentmaterial is fluidly coupled to the sample.
 27. The method of claim 26,wherein the pressure is modulated by placing the absorbent material at ajunction between the at least one channel and the at least one well,whereby the absorbent material absorbs material in the well, therebymodulating flow of the material into the well and regulating the flowrate of said material.
 28. The method of claim 24, wherein the absorbentmaterial is selected from: a solid material, a porous material, a gel, apolymer, a thermoplastic polymer, a porous plastic, and a polyolefinresin.
 29. The method of claim 24, wherein the absorbent material isselected from: paper, dried polyacrylamide, dry sephadex, a dextranparticle, a polyethylene particle, a polypropylene particle, a highmolecular weight polyethylene particle, a polyvinylidene fluorideparticle, an ethylene-vinyl acetate particle, a polytetrafluoroethyleneparticle, a stryene-acrylonitrile particle, a polysulfone particle, apolycarbonate particle, a polyhthalate particle, and combinationsthereof.
 30. The method of claim 24, wherein the material furthercomprises a surfactant.
 31. The method of claim 26, wherein the at leastone well has an upper edge, and wherein the absorbent material projectsbeyond the upper edge.
 32. The method of claim 26, wherein the absorbentmaterial is positioned entirely within the at least one well.
 33. Amethod for determining velocity of a particle, comprising: (i) injectinga particle into a microfluidic channel, (ii) transporting the particlethrough the channel, (iii) detecting a signal from the particle for aperiod of time, the signal having an amplitude corresponding to a numberof particles, and a duration; and, (iv) measuring the duration andamplitude of the signal, thereby determining the velocity of theparticle.
 34. The method of claim 33, wherein the signal is selectedfrom optical, electrochemical, fluorescent and thermal.
 35. The methodof claim 33, wherein said detecting is performed by monitoringfluorescence, phosphorescence, radioactivity, pH, charge, absorbance,luminescence, or magnetism.
 36. The method of claim 33, wherein thevelocity is controlled by electrokinetic injection or withdrawal of amaterial downstream of the particle, wherein the electrokineticinjection of the material modulates pressure downstream of the particle,thereby modulating the flow rate of the particle.
 37. The method ofclaim 33, wherein the velocity is controlled by an absorbent materialdownstream of the particle wherein the absorbent material is in fluidiccommunication with the particle.
 38. A microfluidic device comprising:(i) a body structure having two or more intersecting channels fabricatedtherein; (ii) at least one well fluidly coupled to at least one of theat least two intersecting channels; and, (iii) at least one wickcomprising an absorbent material, wherein the wick is in fluidiccommunication with at least one of the at least two intersectingchannels.
 39. The device of claim 38, wherein the wick is positioned ata junction between the at least one well and at least one of the two ormore intersecting channels.
 40. The device of claim 38, wherein the atleast one well is a waste well in fluidic communication with at leastone of the two or more intersecting channels.
 41. The device of claim38, wherein the absorbent material is selected from: a solid material, aporous material, a gel, a polymer, a high salt fluid, a thermoplasticpolymer, a porous plastic material, and a polyolefin resin.
 42. Thedevice of claim 38, wherein the absorbent material is selected from:paper, dried polyacrylamide, dry sephadex, a dextran particle, apolyethylene particle, a polypropylene particle, a high molecular weightpolyethylene particle, a polyvinylidene fluoride particle, anethylene-vinyl acetate particle, a polytetrafluoroethylene particle, astryene-acrylonitrile particle, a polysulfone particle, a polycarbonateparticle, a polyhthalate particle, and combinations thereof
 43. Themethod of claim 38, wherein the material further comprises a surfactant.44. The device of claim 38, wherein the at least one well has an upperedge and wherein the absorbent material projects beyond the upper edge.45. The device of claim 38, wherein the absorbent material is positionedentirely within the at least one well.
 46. A microfluidic systemcomprising (i) a body having two or more intersecting channelsfabricated therein, (ii) an electrokinetic control element operablycoupled to at least one of the at least two channels, wherein duringoperation of the microfluidic system, the electrokinetic control elementapplies an electrical current within at least one of the at least twochannels, thereby modulating flow of materials within the at least onechannel; (iii) a non-electrokinetic fluid pressure control elementoperably coupled to at least one of the at least two channels, whichnon-electrokinetic fluid pressure control element modulates fluid flowin the at least one channel during operation of the microfluidic system.47. The system of claim 46, wherein the non-electrokinetic fluidpressure control element comprises an absorbent material, whichabsorbent material is in fluid communication with the at least one ofthe at least two channels.
 48. The system of claim 46, furthercomprising a computer operably linked to the system, which computercontrols one or more of the following: the electrokinetic controlelement, the non-electrokinetic fluid pressure control element,monitoring of flow rates, and detection of the materials.
 49. The systemof claim 46, wherein the electrokinetic control element comprises anelectrode.
 50. The system of claim 46, the system operably linked to acomputer comprising software, which software controls simultaneous orsequential monitoring of flow rates.